Increased or decreased matrix remodelling is involved in many diseases and pathological processes, including cancer, angiogenesis, restenosis, arteriosclerosis and other vascular remodelling processes, vascular complications of diabetes such as retinopathy and nephropathy. Dysregulation of remodelling processes frequently causes problems or might be even essential in the development of the particular disease. Matrix remodelling plays a key role in invasion and metastasis of tumor cells, cartilage degradation in rheumatoid arthritis and vascular remodelling. Proteolytic enzymes belonging to the plasmin-system and the matrix-metalloprotease system are main actors in the various remodelling processes. Consequently measuring these components or influencing their activity are potential targets for diagnostic or therapeutic method development.
Vascular complications and renal disease are frequent complications of e.g. diabetes mellitus. Early renal disease is accompanied by leakage of protein into the urine (microalbuminuria) which is used as a diagnostic marker for this complication. Microalbuminuria appears to be preceded by widespread endothelial dysfunction, therefore much attention has been devoted to the study of the possible use of circulating endothelium-derived molecules as markers of early stage diabetic kidney failure. Local proteolytic activity is involved in micro-vascular damage leading to kidney failure and measurement of the local level or activity of certain proteolytic enzymes or their inhibitors might be indicative of occurring damage to microvascular integrity. Some data have been presented suggesting that increased levels of matrix metalloproteases, one group of proteolytic enzymes thought to be involved in formation of vascular damage, occur in peripheral blood possibly as a representation of local levels in the kidney.
For the above, reference is inter alia made to I. Massova et al., FASEB J. 12, 1075-1095 (1998); P. Primakoff et al., Trends Genet. 16, 83-87 (2000); and B. L. Tang and W. Hong, FEBS Lett. 445, 223-225 (1999). However, none of these references discloses or suggests to determine proteolytic enzymes in urine samples.
Generally, the measurement of proteolytic enzymes in periferal blood, serum or plasma is cumbersome, as it involves the use of blood samples, which have to be collected from the patient by a docter or a skilled medical technician. This may put a great deal of strain on the patient, not only because of the invasive techniques required to collect the blood sample, but also because this will usually involve a visit to a clinic or a hospital. In addition blood, serum or plasma contain numerous compounds potentially interfering with the measurement of proteolytic enzymes requiring carefully developed procedures which are prone to complications. In urine hardly any interfering substances occur, thus making reliable measurement of proteases much more simple.
Lenz et al., Journal of the American Society of Nephrology, March 2000, Vol. 11, No. 3 review the role of matrix metalloproteinases in renal development and disease, including non-inflammatory glomerular diseases such as diabetic nephropathy.
However, in doing so, Lenz et al. refer either to in vitro studies involving the measurement of MMP-expression and -activity at the cellular level (e.g. in mesangial cells), to studies involving the use of kidney biopsies, or to studies involving the measurement of expression of MMP-encoding mRNAs. Thus, this reference does not suggest to measure MMPs in urine, and also does not teach that the levels of proteolytic enzymes in urine can be used as diagnostic markers for (early) kidney damage associated with diabetes (mellitus).
Furthermore, with reference to the studies mentioned in their review, Lenz et al. teach that, contrary to inflammatory glomerular diseases such as glomerulonephritis, which are generally associated with increased expression and activity of MMPs, MMP-expression and MMP-activity are in fact decreased in non-inflammatory glomerular diseases such as diabetic nephropathy. In this respect, it should be noted that according to the invention, early renal damage associated with diabetes was found to be associated with a marked increase in the levels/activity of—for example—MMP-2 and MMP-9 in urine.
Senatorksi et al., Res. Exp. Med (Berl) 1998 December; 198(4):199-206, describe a study in which urine activity of cathepsin B and collagenase as well as urine excretion of TGF-β1 and fibronectin were measured in patients suffering from membranous glomerulonephritis.
However, as stated in the first paragraph of the section entitled “Materials and methods” on page 201, this study specifically excluded patients with diabetes. Thus, this reference teaches the skilled person nothing on the levels of proteolytic enzymes in the urine of patients suffering from diabetes. In particular, this reference does not teach that the levels of proteolytic enzymes in the urine of a diabetes patient can be used as diagnostic markers for (early) kidney damage.
In addition, Senatorksi et al. generally refer to the measurement of “collagenases” in urine, in which the term “collagenases” is apparently used generically to indicate any and all enzymes from the (very extensive) entire group/family of enzymes that may degrade native collagen. This is confirmed by the fact that the fluorometric technique used by Senatorksi to determine said “collagenases” is not very specific (e.g. for individual collagenases or MMPs).
Also, two of the most important markers in urine used according to the present invention, MMP-2 and MMP-9, are not “collagenases” but “gelatinases” (compare the discussion of MMP-2 and MMP-9 in the review article by Lenz et al., mentioned above).
In the invention, it has now been found that the presence and/or the levels of certain proteases in the urine of a subject may be used as a diagnostic and/or prognostic parameter for renal disorders, and in particular for (early) renal damage associated with diabetes mellitus.
In particular, in the invention, it has been found that the presence and/or the levels of certain proteases in urine may be indicative for renal damage which is not—or not yet—detectable by the known method of determining the leakage of protein in the urine. Thus, the method of the invention is not only more reliable than determining (the extent of) microalbuminuria, but may also provide valuable information to the clinician even before any damage to the kidneys has already progressed to such a stage that microalbuminuria occurs.
Also, in the invention, it has surprisingly been found that the presence and/or the levels of said proteases in urine may be used as reliable markers for (early) renal damage which is not—or not yet—detectable by increased levels of matrix metalloproteases in peripheral blood. Thus, besides putting less strain on the patient and generally being more convenient, the method of the invention is also more reliable than measuring the amount of proteases in blood or plasma, allowing for earlier detection of possible renal damage, in particular in diabetic patients.
It should however be noted that the invention is not limited to (early) detection of renal damage in diabetes or other diseases with kidney failure as complication, but can also be employed for monitoring or diagnosis of remodelling of the peritoneal membrane. Remodelling of the peritoneal membrane is a frequent problem in patients undergoing continuous ambulatory peritoneal dialysis (CAPD). For instance, one of the further (non-limiting) applications of the invention that is envisaged is to monitor adequacy of dialysis in such patients.